Assessment of properties of gluten‐based edible film formulated with beeswax and DATEM for hamburger bread coating

Abstract Using edible films and coatings is one of the effective methods of improving the quality of bread. The aim of the present work was the development of gluten‐based films containing lipids to be applied as bread coating, intending to improve quality and delay staleness. In this study, two types of lipids including beeswax and DATEM (diacetyl tartaric ester monoglycerides) were incorporated into gluten film at different levels. The findings showed that inserting both lipids together into gluten for film preparation, weakened the developed films in terms of mechanical and moisture barrier properties. Adding DATEM to the gluten film formulae decreased the elongation at the break and the tensile strength of the film. Using gluten‐beeswax coatings for hamburger bread, compared to gluten‐DATEM coatings, indicated a significant decrease in the hardness and staling feature. Moreover, applying sorbate as a preservative along with the solvents used in the film preparation prevented the growth of mold during the bread shelf life. In conclusion, the findings in this study indicated that the type and levels of lipids added to the edible gluten‐based films and coatings affected the film properties and coated hamburger bread quality, significantly.

increasing the shelf life of bread and consumer acceptance is applying various types of edible films and coatings on the bread surface.
Edible films and coatings are thin layers of edible materials that are placed on the surface of food and are considered as one of the basic ways to control physiological, microbial, and physicochemical changes in food. Edible coatings and films refer to different ideas.
While edible coatings are generated directly on the surface of the food products, edible films are thin, solid layers that can be applied to wrap food . Polysaccharides, lipids, and proteins are the three kinds of ingredients that can be employed to create edible films and coatings. The film-forming solutions may also contain other components, such as plasticizers, to increase their mechanical or structural stability (Valencia-Chamorro et al., 2011).
The main purpose of using edible films and coatings is to prevent the transfer of moisture and oxygen, preserve aromatic substances, flavoring compounds, or oils, improve the quality and appearance and increase the shelf life of food (Silva-Weiss et al., 2013).
Very high molecular weight, high complexity, and diversity of components are the characteristics of wheat gluten protein that can be used to make edible films and coatings with practical properties. Film formation from wheat gluten solutions has been widely studied (Hernandez-Munoz et al., 2004;Madhu et al., 2020;Shen et al., 2022;Tanada-Palmu & Grosso, 2003). This process requires the formation and drying of a thin layer of the protein matrix (Wittaya, 2012). The barrier properties of gluten films, such as moisture permeability, can be improved by incorporating a lipid material into their structure (Shen et al., 2022). Diacetyl tartaric ester monoglycerides (DATEM) and beeswax, because of their high moisture retention capacity as hydrophobic substances and their good appearance, are the most effective lipid materials for improving the barrier properties of gluten-based films (Hammam, 2019). DATAM is an emulsifier that is mainly used in the bakery industry. It is a combination of complex glycerin esters in which one or more hydroxyl groups are esterified with diacetyl tartaric acid and mono or diglyceride. Combining wheat gluten protein with DATEM could reduce water vapor permeability, increase tensile strength, and maintain transparency (Bourtoom, 2009).
Although previous works have demonstrated the enhancing effects of various types of gluten-based films or coatings for applying to bakery products, investigation regarding the role of lipids, DATEM and/or beeswax, in improving the properties of the gluten-based films or coatings is not performed yet. This work thus aimed to study the optimum formulation of edible protein-lipid film and coating and to investigate their use in order to increase the quality of hamburger bread.

| Materials
Commercial-type wheat gluten, with the parameters of 4.33% moisture and 80.91% protein, and hamburger bread were purchased from Nanavaran Co. (Tehran, Iran). DATEM was supplied by the enzyme India Company (Karnataka, India). The Beeswax was provided by Fluka company (Buchs, Switzerland). All other chemicals used in this study were obtained from Merck (Darmstadt, Germany).

| Film preparation
A suspension of 12.5 g gluten, 20 ml distilled water, and 0.03 g sodium sulfite was prepared. 32 mL ethanol was added after 15 min and the solution was stirred for 10 min at 40°C using a magnetic stirrer (RH-B2, IKA, Germany). After the addition of 2 ml glycerol, as a plasticizer, the pH of the mixture was adjusted to 5 and the solution was stirred for 10 min using a magnetic stirrer. Then 3 g lipid, including DATEM and/or Beeswax (see Table 1), for each treatment was dissolved and the mixture was heated over the melting point of lipids. Due to the high humidity of the bread surfaces, 0.18 g sorbate (0.01 of the dry matter of the film coating) was added to the solution in this step, in order to prevent mold growth. The final solution was homogenized for 4 min using a homogenizer. Then, the solution was placed on a glass plate (with a thickness of 120 microns) at 25°C and 50% relative humidity for 20 h to be completely dry. Dried films were peeled off the glass petri dishes slowly and maintained in a desiccator at 25°C and 50% relative humidity for 48 h before usage. Water solubility, water vapor permeability, and mechanical properties of the prepared films were measured to select the optimal film.

| Mechanical properties
Mechanical properties of the films, including tensile strength and elongation at break, were determined according to Mohammadi, Azizi, and Zoghi et al. (2020) with some modifications using an Instron Universal Testing Machine (BZ2.5/TH1S, Zwick, Germany) according to the ASTM-D882-02. The films were cut into 1.4 × 6 cm pieces and equilibrated at 25°C and 50% relative humidity. Then fixed between the two jaws with an initial distance of 40 mm. Tensile strength was obtained by dividing the peak load in Newton (N) by the cross-sectional area of the initial film pieces. Elongation at break was calculated by the percentage change in length of the film pieces from 40 mm of the original distance between the jaws.

| Water solubility
Water solubility was determined based on the method of Mohammadi et al. (2020). Three circles with a diameter of 2 cm were cut from each film, then the pieces of film were heated in an oven (L3456J, Iran Khodsaz, Iran) at 100°C for 24 h to a constant weight.
Then, the pieces of films were immersed in containers containing 50 ml of distilled water and 0.02% (v/w) Sodium azide, to prevent the growth of microorganisms, for 24 h at room temperature on a magnetic stirrer (RH-B2, IKA, Germany). The films were then filtered to obtain undissolved films and dried at 100°C to reach a constant weight to obtain the final dry weight of the film. By reducing this weight from the weight of the initial dry matter, the weight of the dry matter dissolved in water and its percentage was obtained.

| Water vapor permeability
This value was determined using the standard ASTM-96-80 method with some modifications. Circular cups with an inner diameter of 3.5 cm, an outer diameter of 5.5 cm, and a depth of 3 cm containing 8 ml of distilled water were sealed by the films with clips and grease.
Each cup was placed inside a desiccator with silica gel at 25°C and was weighed every 12 h (using a digital scale with a precision of 0.0001). Water vapor passing through the film and absorbed by the silica gel is obtained by increasing the weight of the silica gel. The relative humidity inside the cup was 100% and the outside of the cup was 0%, which caused a slight partial pressure of 32.23 mmHg between the outside and inside of the cup.
The water vapor permeability was calculated according to the following equation: Where ΔW is weight loss per cup (g); X is film thickness (mm); A is the area of exposure to the cup (9.16 cm 2 ); T is time (s); and Δp is the pressure difference on both sides of the film (mmHg).

| Color measurement
The color values of films in terms of L* (lightness), a* (redness), and b* (yellowness) were measured using a colorimeter (Cdorflex, USA).
Films were placed in desiccators at 25°C and 50% relative humidity for 48 h before measurement (Hernandez-Munoz et al., 2004).
Triplicate measurements of color were conducted for each film.

| Coating of bread samples
The prepared solution for film production was sprayed onto the whole surface of the fresh hamburger bread samples using a spray device (WALMEC, Asturo///EC, Italy). For this purpose, a 30 ml solution was sprayed evenly on each bread surface with a pressure of 2 bar, and a constant distance of 20 cm. Then the bread samples were immediately placed in an oven (L3456J, Iran Khodsaz, Iran) at 60°C for 30 min to evaporate the solvents and form a coating (Bravin et al., 2006).

| Hardness evaluation
Some of the bread samples were placed in polyethylene bags and kept at room temperature, and others were placed without polyethylene bags at 50% relative humidity and 25°C inside a humidifier (IKH.RH, Iran Khodsaz, Iran). The hardness of bread samples was analyzed by a bread hardness tester (H50KS, Hounsfield, UK) according to AACC (2000) No. 74-09 in the first, third, and fifth days of storage time for samples with polyethylene bags, and in the first, second, and third days of storage time for samples without polyethylene bags.

| Sensory evaluation
The staling feature of samples was assessed according to AACC (2000) No. 74-30, after 1, 3, and 5 days of storage time in Polyethylene bags at room temperature. For this purpose, 25 trained panelists were asked to rate the bread samples in the ranks of 1 to 6 in terms of staleness. So, the freshest bread was given the rank of 6 and the stalest bread was given the rank of 1. The samples were identified with random three-digit numbers.

| Evaluation of the appearance of coated bread in terms of mold growth
Samples in polyethylene bags were evaluated visually for mold growth on the first, third, and fifth day of storage time at 25°C (Plessas et al., 2008).

| Statistical analyses
The data were analyzed by one-way analysis of variance (ANOVA) and comparison of the mean of data with Tukey's test. p values <.05 were considered statistically significant. All tests were carried out three times and analyzed using SPSS 16 statistical package (SPSS Inc.). Friedman test was used to evaluate the results of the sensory assessment of bread samples.  (Tosif et al., 2021). Adding beeswax to the gluten-based film weakens the film and increases its elasticity, which is due to the diffusion of beeswax particles inside the gluten network and reducing the strength of this network.

| Mechanical properties of the film
However, due to the reaction of DATEM with gluten, it does not play a significant role in the resistance of the film to tension, and the tensile strength is significantly reduced due to the strength of the gluten network by the DATEM (Hammam, 2019). As can be seen in Table 2, there is a significant difference in tensile strength between all treatments. The highest tensile strength was observed in treatment 1 (8.96) and followed by treatments 2 and 3. In treatment 4, the lowest tensile strength was seen (3.51). So, in general, it can be said that DATEM has reduced the tensile strength in the gluten-based film to some extent, but beeswax causes a sharp decrease in tensile strength.
It has been reported that increasing beeswax supplementation with film formation solution led to decreasing the tensile strength and increasing the elongation of the gluten-based films (Hammam, 2019). In general, the higher the lipid concentration, the lower the tensile strength value, which is in line with many previously reported studies (Shen et al., 2022;Zhang et al., 2018). This result can be attributed to the non-polymeric nature of lipids, which inhibits their ability to form cohesive films, leading to the film's heterogeneous structure (Kowalczyk et al., 2016). Treatment 4 is an exception; The reason for this can be expressed by the negative effect of DATEM and beeswax in this ratio (50/50) on the gluten network.
The elongation at break decreased with the addition of DATEM (See Table 2). This decrease may be caused by solid particles that can cause the formation of a rigid dispersed phase in the film, in turn leading to decreased flexibility of the film (Shen et al., 2022). In contrast, the elongation at break increased with the incorporation of beeswax, suggesting that beeswax has a strong plasticizing effect on the films, causing the network structure of the film to be discontinuous (Valizadeh et al., 2019). Therefore, the highest amount of elongation at break was observed in treatment 6, which contains just beeswax. Similar results have also been previously observed (Nurul Syahida et al., 2020;Shen et al., 2022).

| Water solubility of the film
Increasing lipids in gluten-based films reduce the solubility of these films in water and this is due to the hydrophobicity of lipids (Shen et al., 2022). However, due to DATEM's hydrophilic nature, it reduces gluten-based film solubility less than hydrophobic lipids (Avramescu et al., 2020). As shown in Table 2, in treatment 1, which contains gluten without lipids, the solubility is 20.60%. By adding DATEM, the solubility is reduced so that in treatment 2, which contains 100% DATEM, the solubility is 16.07%. By decreasing the percentage of DATEM and increasing the percentage of beeswax in the formula, the solubility decreased more, and finally, the lowest amount (13.33%) was observed in treatment 6 with 100% beeswax.
Therefore, it can be concluded that the lipid-free gluten-based film had the highest water solubility, and the gluten-based film containing just beeswax had the least water solubility. This result is con-

| Water vapor permeability of the film
The addition of lipid components up to 20 g per 100 g of dry matter of the gluten-based film, leads to a sharp decrease in the water vapor permeability of the film, and this is due to the presence of many non-polar groups and their hydrophobicity that prevents the transfer of water molecules through the film (Shen et al., 2022). Table 2, treatment 1 (lipid-free) had the highest water vapor permeability, treatment 6 (just beeswax) had the lowest water vapor permeability, and the rest of the treatments are in the middle. Beeswax is a hydrophobic lipid, and could thus effectively reject moisture and reduce hydrophilic interactions; thus, it could significantly decrease the water vapor permeability values of the glutenbased films. In general, waxes normally are comprised of esters of fatty acids and long-chain alcohols (Jannin & Cuppok, 2013). They do not possess any polar constituents or hydrophilic parts, indicating that they cannot interact with water. Therefore, waxes have very low water vapor permeability (Chen et al., 2021).

| Color analysis of the film
Color is an important organoleptic parameter, and the success of coating it is represented by the final product color (Avramescu et al., 2020). The results obtained from the film color measurement (Table 3)

| Hardness evaluation of coated bread
According to the results, treatments 2 and 6 (see Table 1) were selected as the optimum formula for coating bread samples. Because treatments 6 and 2 had the best characteristics in terms of water vapor permeability and mechanical properties, respectively. So, other experiments were done using a control sample (bread without coating), and bread samples with coating treatments 2 and 6.
Based on the data in Table 4, the hardness of bread coated with treatment 6 (just beeswax) on the 1st, 3rd, and 5th days of storage, in polyethylene bags, was significantly different from bread coated with treatment 2 (just DATEM) and bread without coating. Bread coated with treatment 6 showed the least hardness, and then bread coated with treatment 2 was placed, and the highest hardness was related to bread without coating. According to Table 5, the same result was obtained for the hardness of coated and uncoated bread samples without the use of polyethylene bags at 50% relative humidity. Since treatment 6 had lower water vapor permeability compared to treatment 2, these results were expected. The reason for this result is that less moisture was removed from the coated bread samples than from the uncoated ones and therefore, they become softer.
One of the most crucial elements affecting bread hardness is the moisture content of the crumb. Reducing moisture enhances the hydrogen bonding between starch filaments and the development of links between protein and starch, which in turn promotes hardness because water can act as a softener in bread (Silva et al., 2016). Similar results have been obtained in this field, which shows the positive role of these coatings in reducing hardness (Balaguer et al., 2013;Chen et al., 2021;Heras-Mozos et al., 2019). Bravin et al. (2006)

| Sensory assessment of coated bread
Considering the mean scores of sensory tests of coated and uncoated bread samples on days 1, 3, and 5 of storage (Table 4), it can be concluded that on the first day there was no significant difference between the three samples; and on the third and fifth days, there was no significant difference between the two coated bread samples, but there was a significant difference between them and bread without coating. Significant differences between coated bread samples and uncoated bread are consistent with the hardness evaluation test results and show less staleness in coated bread samples. Staleness is a process that reduces the acceptability of baking industry products by consumers and includes changes in the core of bread.

TA B L E 3 Color analysis of gluten-based edible films
Generally, staleness is characterized by leathery bread crust, hardening of bread core, decrease in moisture and taste, and decrease in freshness in the product (Silva et al., 2016). During the bread's storage period, complicated molecular structure changes cause the staling phenomena to take place. Bread's freshness is negatively impacted by water loss during the shelf life, which causes earlier staling (Kim & Yoo, 2020). Therefore, using edible coatings could help delay staling in hamburger bread. Chen et al. (2021) studied a wide range of edible coating ingredients, including beeswax, gum, starch, protein, and cellulose derivatives to control bread staling. According to their results, all wax coatings could decrease bread moisture loss, crumb hardening, and retard bread staling significantly (Chen et al., 2021).

| Evaluation of mold growth in coated bread
Due to the addition of sorbate as a preservative and the use of showed the first symptoms of mold growth on day 5, but in the case of film-coated with ethylene-vinyl alcohol copolymer or polyethylene containing 0.5% of garlic extract, mold growth began on days 9 and 12, respectively.

| CON CLUS IONS
In this work, a gluten-based film containing lipids, beeswax and/ or DATEM, was successfully applied for the coating of hamburger bread in order to reduce staling process and improve

ACK N OWLED G M ENTS
The authors would like to thank the National Nutrition and Food Technology Research Institute (Iran, Tehran) for the provision of the equipment required for measurement.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

TA B L E 4
Hardness and staling feature of coated hamburger bread samples on the first, third, and fifth day of storage time in polyethylene bags

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article.

E TH I C S S TATEM ENT
We declare no ethical issue related to this article.